Conotruncal defects (CTDs) comprise 36% of all congenital heart defects and carry significant morbidity. Although their etiology is poorly defined, data suggest that complex genetic mechanisms contribute to their etiology. This Program will define the genetic basis of CTDs. Molecular evaluation of genetic syndromes with CTDs have provided valuable insight into their genetic basis. In particular, studies on the 22q11.2 deletion syndrome defined a large CTD population, identified genes (e.g. TBX1) and developmental pathways contributing to cardiac development and disease. To continue this work, Project 1 will identify genetic modifiers of CTDs in an exceptional, large 22q11.2 deleted patient cohort using genome wide approaches. In Project 2, genome wide studies in a unique, large non-syndromic patient CTD cohort will be completed to identify both case (inherited) and maternal genetic effects. Discoveries made in one patient cohort will be examined for significance in the other. Mouse models based on 22q11 DS will be used to elucidate developmental pathways critical to conotruncal morphogenesis. Genes and developmental pathways described in the mouse models will be examined for disease associated genetic variants in each of the two patient cohorts, and discoveries in Projects 1 and 2 will in turn be examined in the mouse for expression pattern and placement in key developmental pathways. Candidate genes from these studies will be subject to deep sequencing to identify the full range of disease related genetic variants. The proposed studies are highly interactive, leverage unique patient cohorts and mouse models, build upon long standing collaborations, and test the hypotheses that: (1) the 22q11.2 deleted cohort will serve to unmask genetic risk factors for the characteristic cardiac defects, (2) these risk factors apply to the non-syndromic cardiac cohort, and (3) critical developmental pathways can be elucidated in the mouse whose gene members are disease- related in humans. These studies wilt greatly expand our understanding of the genetic basis of CTDs, and will promote the development of novel therapeutic and preventive strategies. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the genetic causes of congenital heart defects. Project 1 is to find genetic modifiers of heart defects in 22q11 DS. Project 2 is to find genetic risk factors for non-syndromic conotruncal heart defects and Project 3 will use mouse models to help determine which of the candidates identified in Projects 1 and 2 function in cardiac development. OVERALL PROGRAM: The goal of the Program is to identify genetic modifiers of 22q11 deletion syndrome. The proposed work extends similar studies supported by an R01 consortium grant to the group. The investigators have a strong record of collaboration and are leaders in the genetic analysis of 22q11DS. A major strength of the program is the availability of 22q11DS patient DNA samples and diagnostic information that is unique in the world. Central focus on CTDs is solidly evident in all projects. The team is composed of experienced, talented investigators, with strong environments. The established collaboration between Drs Goldmuntz and Emmanuel also is a strength. Sharing data sets and the aid in using animal modeling to validate isolated genes has potential. The program is highly integrated throughout and extremely synergistic. Project and Core infrastructure support extensive genomic analyses and platforms. Preliminary studies have already identified several copy number and SNP variants by GWAS. Overall it seems likely that statistically significant associations will be established that could represent genetic modifiers of 22q11DS or causes of nonsyndromic CTM. Proposed animal models in Project 3 are designed to provide necessary functional validation of identified variants. The overall impact of the study is diminished in that 22q11DS patient samples have already been subjected to extensive genomic analyses and the primary determinant of the 22q11 phenotype is already known. In addition multiple locations put a strain on interactions and communication mechanisms not well addressed in the Administrative core given the multiple locations. Details on meetings, and other forms of communication would strengthen enthusiasm 1.Significance of the overall program and its potential to advance scientific knowledge: Strengths * Conotruncal malformations are a significant cause of congenital heart disease and 22q11DS is a leading contributing cause to these types of malformations. * Multiple genetic and genomic approaches will likely identify loci of possible genetic modifiers associated with 22q11DS conotruncal malformations. * These 22q11DS modifiers identified in Project 1 may also be associated with nonsyndromic conotuncal malformations. The genetic basis of nonsyndromic CTM will be examined in Project 2. Therefore there is strong synergy in these projects. * Specific Tbx1 interacting pathways will be examined. Tbx1 is the main causative gene in the 22q11 locus associated with conotruncal malformations. These studies taken together with the CTM genetic association studies may provide new mechanistic data on the origins of CTM. * Validation of data sets from Projects 1 and 2 in an animal model is a strength Weaknesses * The primary genetic lesion of 22q11DS is already known and extensive genomic analyses of these patients have already performed by this group and others. * In preliminary studies, CNV and GWAS studies have already identified candidate loci of 22q11DS modifier genes and variants associated with CTM. The proposed work in large part provides additional validation, replication and numbers for increased statistical power of these analyses. * Tying all molecules identified in this work to TBX1 and associated pathways is unlikely. 2. Investigator(s) (Qualifications of the PIs in Program Leadership and/or in Directing Individual Projects or Cores: Strengths * Dr. Morrow is well-qualified to oversee the program and has strong administrative experience. * The research team has worked together for a long time and has led the field in terms of genetic and mechanistic analyses of 22q11DS and Tbx1-related CTM. * There are skilled and experienced key personnel for statistical, genetic and bioinformatic support. Weaknesses * There may be some overlap in efforts by having genetic and bioinformatic analyses performed at the two separate locations. * No central location 3. Innovation (Applying Novel Concepts and Innovative Approaches to the Overall Program and Collaborations): Strengths * The collection of patient DNA samples and diagnostic data to be used in the proposed studies is unique to this group. * State of the art genomic approaches are proposed and new technologies will be considered as they become available. * Somewhat innovative by trying to use mouse expression analysis to confirm putative relevance to CTDs Weaknesses * Extensive genomic and genetic analyses have already been performed using the 22q11DS and nonsyndromic CTM patient samples available. Potential SNP and copy number variants have already been identified by GWAS approaches in preliminary studies. * Using TBX1 as a central hub may not be in best interest of discovery * Difficult to push the envelope when locations are scattered. 4. Approach (Overall Design of the Program Project and Adequacy and Quality of the Experimental Approaches Proposed in the Projects): Strengths * The overall approach in the program is highly synergistic and the results generated in each project will be incorporated into the other projects. Together, the integration of the projects will enhance the potential for identification of significant variants in 22q11DS and CTM with validation in animal models that have implications for the development of cardiac malformations. Therefore the program is very focused and the projects are dependent on one another in terms of extracting the maximal information from the data. * The approach takes full advantage of multiple technologies and platforms for the proposed genomic studies. The research team has experience in this area and extensive genetic data will be generated and analyzed. * Solid approaches defined by projects as would be expected from established successful investigators Weaknesses * In each project, multiple types of CTM are considered as a single entity. Specific types of malformations may have distinct molecular and developmental origins, but this is not taken into account in the experimental design. * Genomic studies in Projects 1 and 2 will be performed at different sites, using different platforms. The integration of these data would likely be more effective if performed using the same technology in the same facility. In some cases the specific platform or technology to be used is not identified, but several possibilities are mentioned as an approach yet to be determined. This was not viewed as a significant weakness, since the group has extensive experience in this area. * Utilization of mRNA expression as sole measure of interactive relationships (Project 3) is circumstantial. 5. Environment (Scientific, Organizational/Institutional, and Administrative Environment of the Program): Strengths * The environment at Einstein and CHOP is outstanding for the proposed studies. * The organization and administrative structure of the program are strong. Weaknesses * It is not clear why some of the genotyping will be done at Einstein using one platform (Project 1) and comparable genotyping (Project 2) will be done at CHOP using a different platform. * Integration of all locations is not well described. COMPONENT PROJECTS: Project 1 - Genetic Mechanisms for Conotruncal Heart Malformations Description (as provided by applicant): The 22q11.2 deletion syndrome (22q11DS) is a congenital anomaly disorder occurring in 1/4,000 live births. It is occurs due to a 3Mb deletion of chromosome 22 at band q11.2. Approximately 70% of children with 22q11DS have congenital heart disease of the conotruncal type (CTD), while 30% have no cardiac malformations as determined by echocardiography. It is likely that many factors such as stochastic events, environmental exposures and specific genes or gene pathways play a role in modifying the phenotype. This project proposes to explore and discover genetic factors that underlie the differences between 22q11DS patients with and those without a CTD. To identify genetic modifiers of CTDs, we have performed a genome- wide CNV and SNP association study of 650 22q11DS subjects using Affymetrix 6.0 arrays. We have found rare CNVs of biological interest. To enhance the significance of the CNV findings and SNP associations, we propose to expand the study of already identified rare and common CNVs by expanding the cohort to include a total of 1,150 22q11 DS patients. Thus, we will run 500 additional Affymetrix 6.0 microarrays. A GWAS will be performed on the entire cohort for SNP based modifier loci. We will compare our candidate loci with results from Project 2. Project 2 will perform a replication of their previous GWAS of non-syndromic CTD patients and normal controls, by adding 700 additional cases and additional controls to identify risk factors for CTDs. We will compare the two datasets to identify loci in common, requiring selected re-genotyping after imputation, as the studies used different platforms (lllumina versus Affymetrix). To confirm that the loci identified are CTD candidate genes, in situ hybridization on mouse embryos will be performed. It is possible that genes in the genetic pathway of TBX1, a gene in the deleted interval, can serve as modifiers. TBX1 encodes a T-box transcription factor, which in mouse models is responsible for many of the features of the 22q11DS. Therefore, 22q11DS mouse models will be evaluated to determine if the genes are in a shared pathway, such as the Tbxl pathway (Project 3). Genetic pathways of relevance that are identified by Project 3 will be examined in the CNVs and SNP loci identified in this Project. Targeted re-sequencing will be performed in an attempt to identify causative genes that modify the CTD phenotype in 22q11DS individuals. The overall outcome of the combined human and mouse program will enable us to systematically explore genetic risk factors for CTDs. This project will expand our understanding of the genetic basis of CTDs, and provide a first step towards the development of novel therapeutic and preventive strategies. PUBLIC HEALTH RELEVANCE: Congenital heart defects of the conotruncal type occur in 70% of 22q11 DS patients while 30% have no cardiovascular anomalies. Our goal is to find genetic modifiers in our cohort of 1,150 subjects taking genome wide approaches. This work will uncover new genes that may alter risk of heart defects for 22q11 DS and non-syndromic patients. This will be tested by comparing our data to data from Project 2 derived from non-syndromic conotruncal defect subjects. D